Structural and biological analysis of the metal sites of Escherichia coli hydrogenase accessory protein HypB.
The [NiFe]-hydrogenase protein produced by many types of bacteria contains a dinuclear metal center that is required for enzymatic activity. Assembly of this metal cluster involves the coordinated activity of a number of helper proteins including the accessory protein, HypB, which is necessary for Ni(II) incorporation into the hydrogenase proteins. The HypB protein from Escherichia coli has two metal-binding sites, a high-affinity Ni(II) site that includes ligands from the N-terminal domain and a low-affinity metal site located within the C-terminal GTPase domain. In order to determine the physiological relevance of the two separate sites, hydrogenase production was assessed in strains of E. coli expressing wild-type HypB, the isolated GTPase domain, or site-directed mutants of metal-binding residues. These experiments demonstrate that both metal sites of HypB are critical for the maturation of the hydrogenase enzymes in E. coli. X-ray absorption spectroscopy of purified proteins was used to examine the detailed coordination spheres of each nickel-loaded site. In addition, because the low-affinity metal site has a stronger preference for Zn(II) than Ni(II), the ligands and geometry for this metal were also resolved. The results from these experiments are discussed in the context of a mechanism for Ni(II) insertion into the hydrogenase protein.
Dias
Alistair V
AV
Department of Chemistry, University of Toronto, Toronto, Ontario, Canada.
Mulvihill
Cory M
CM
Leach
Michael R
MR
Pickering
Ingrid J
IJ
George
Graham N
GN
Zamble
Deborah B
DB
D015502:Absorptiometry, Photon; D019943:Amino Acid Substitution; D001665:Binding Sites / Q000502:physiology; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006864:Hydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008024:Ligands; D008667:Metalloproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D009532:Nickel / Q000737:chemistry* / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000502:physiology; D015032:Zinc / Q000737:chemistry / Q000378:metabolism
D029968:Escherichia coli Proteins; C490324:HypB protein, E coli; D008024:Ligands; D008667:Metalloproteins; D009532:Nickel; D006864:Hydrogenase; D019204:GTP-Binding Proteins; D015032:Zinc